Protein kinase C γ mutations in the C1B domain cause caspase-3-linked apoptosis in lens epithelial cells through gap junctions
Introduction
Gap junctions are hydrophilic channels and/or hemichannels that allow the passage of small molecules including necessary metabolites, second messengers, ions, and cell death signals from cell to cell (Thompson et al., 2006). Propagation or amplification of cell death signals through open gap junctions results in cell apoptosis (Cusato et al., 2003, Lin et al., 1998, Neijssen et al., 2005). Lens epithelial cells express gap junction proteins, Cx43 and Cx50, while lens fiber cells express Cx50 and Cx46. Fiber cell gap junctions provide a pathway to maintain lens homeostasis through circulating fluxes, and disruption of this gap junctional cell-to-cell communication pathway causes cataractogenesis (Goodenough et al., 1996). We have previously determined that gap junctions, Cx43 and Cx50, are the major targets of activated PKCγ in the lens epithelial cells in culture and in the whole lens (Lin and Takemoto, 2005, Lin et al., 2003a, Lin et al., 2003b, Lin et al., 2004, Zampighi et al., 2005). Loss of control of gap junctions by PKCγ, i.e., no PKCγ phosphorylation of Cx50 in PKCγ knockout lenses, causes PKCγ knockout lenses to be more susceptible to oxidative stress-induced cataracts in the mouse (Lin et al., 2006). Inhibition of gap junctions prevents cell death (Farahani et al., 2005, de Pina-Benabou et al., 2005, Krysko et al., 2005). It is apparent that control of gap junctions is essential for lens cell survival.
PKCγ is primarily found in the central and peripheral nervous systems (Shutoh et al., 2003). It is also found in the eye tissues including retina and lens epithelium and cortex (Correia et al., 2003, Saleh et al., 2001). PKCγ consists of C1 and C2 regulatory domains and C3 and C4 catalytic domains. The C1 domain contains two tandem repeat, Cys-rich subdomains, C1A and C1B. The C1 domain is a diacylglycerol (DAG) binding domain, while calcium binds to the C2 domain. Unlike other conventional PKCs, the C1B subdomain of the PKCγ protein is always exposed and calcium is not required for activation of this enzyme (Ananthanarayanan et al., 2003, Lin et al., 2003a). We have demonstrated that the PKCγ C1B subdomain is oxidized when cells are treated with H2O2 which results in formation of disulfide bonds and activation of the enzyme. This occurs without an elevation in cellular DAG levels. The activated PKCγ phosphorylates Cx43 on Ser368 and this causes disassembly of gap junction plaques and inhibition of gap junction dye transfer activity (Lin and Takemoto, 2005). PKCγ acts as an oxidative stress sensor to prevent the lens from oxidative damage through proper control of gap junctions. It has been reported recently that missense mutations in PKCγ cause the dominant spinocerebellar ataxia type 14 (SCA14), a neurodegenerative disorder with onset age as early as 3 years (Chen et al., 2003, Chen et al., 2005, van de Warrenburg et al., 2003, Stevanin et al., 2004, Yabe et al., 2003, Verbeek et al., 2005, Seki et al., 2005, Alonso et al., 2005, Klebe et al., 2005, Vlak et al., 2006, Fahey et al., 2005). Most of the PKCγ SCA14 mutations occur in the C1B subdomain. Since this is a newly identified neuronal disorder, long term follow-up study is necessary to uncover what PKCγ SCA14 mutations do to cataractogenesis. We previously observed that the PKCγ H101Y mutant is not activated by oxidative stress, e.g. H2O2 (Lin and Takemoto, 2005). But its effect on gap junctions and cell survival remains unclear.
In this paper we study the structure/function effects of three PKCγ C1B mutations on PKCγ enzyme activity, lens gap junction control, and on the induction of apoptosis in N/N1003A lens epithelial cells. This cell line expresses Cx43, Cx50, and PKCγ. All these mutants are expressed against a background of endogenous wild type PKCγ. This is because humans with SCA14 are heterozygous. Thus, this more correctly models the human disease. We demonstrate that cells with PKCγ C1B mutants lacked control of Cx43 and Cx50 gap junctions when exposed to H2O2 and this caused cells to be more susceptible to H2O2-induced, caspase-3-dependent cell apoptosis.
Section snippets
Materials
Antibodies against active caspase-3, PKCγ, connexin 43 (Cx43), and GFP were purchased from BD Biosciences (Palo Alto, CA). Rabbit polyclonal PKCγ phospho T514 antibody was purchased from Abcam (Cambridge, MA). Caspase-3 colorimetric assay kit, polyclonal rabbit anti-phosphoserine, and anti-phospho-S368-Cx43 were purchased from Chemicon (Temicula, CA). Protein A/G PLUS-agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse or anti-rabbit IgG conjugated with HRP,
Modeled structures of the C1B mutants
We chose three mutations in the PKCγ C1B domain (Fig. 1A) to test the structure/function relationship of the PKCγ C1B domain. The predicted three-dimensional structure of PKCγ mutants (H101Y, S119P, and G128D) were modeled based on the secondary structure of PKCγ C1 domain for which NMR experimental structural characterization was available (Xu et al., 1997). WT displays a short helix (V142–V146) and five β-strands (H101–S107, T112–C114, S119–L121, G128–C131, and M136–H139). The
Discussion
We have previously demonstrated that PKCγ serves as an oxidative stress sensor through proper control of gap junctions in the lens (Lin et al., 2003a, Lin et al., 2003b, Lin et al., 2004, Lin and Takemoto, 2005, Zampighi et al., 2005, Lin et al., 2006). We have also observed that the PKCγ H101Y C1B mutant is not activated by oxidative stress, e.g. H2O2 (Lin and Takemoto, 2005). In this current study we have tested three SCA14 PKCγ mutants which have modeled structures indicating disruption of
Acknowledgements
The authors are grateful to K.H. Fischbeck of NINDS/NIH for helpful discussions. We thank Dr John Reddan of Oakland University for the N/N1003A cells, Dr Peggy Zelenka of National Eye Institute for the PKCγ:EGFP vector. This work was supported by National Institutes of Health Grant EY 13421 (to D.J.T.) and by a grant from the National Organization for Rare Disorders (to D.L.). This is publication 06-13-5 from the Kansas Agricultural Experiment Station.
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Spinocerebellar ataxia type 14
2012, Handbook of Clinical NeurologyCitation Excerpt :The effect of these mutations on the function of the protein in vivo is not known. However, computer simulation studies on three missense mutations (Chen et al., 2003) and in vitro experiments (Alonso et al., 2005; Verbeek et al., 2005b; Seki et al., 2005, 2007, 2009; Lin et al., 2007; Asai et al., 2009) suggest that mutant gene products may be less stable than the normal protein, prone to aggregation, and may have abnormal activation patterns, altered substrate specificity and altered activity, result in abnormal dendritic development, cause apoptosis, impair the ubiquitin–proteasome system, and induce endoplasmic reticulum stress. It is speculated that the SCA14 phenotype results from gain of function rather than haploinsufficiency because no chain-terminating mutations have been found and heterozygous PKCγ-null animals are neurologically normal (Abeliovich et al., 1993); a dominant-negative mechanism cannot be ruled out at this time.
Mutant γPKC found in spinocerebellar ataxia type 14 induces aggregate-independent maldevelopment of dendrites in primary cultured Purkinje cells
2009, Neurobiology of DiseaseCitation Excerpt :Since basal activities of mutant γPKC examined in the present study (S119P and G128D) were also increased, these increased activities of mutant γPKC might trigger the maldevelopment of PC dendrites. However, Lin et al. reported that mutant (H101Y, S119P and G128D) γPKC expression reduced the kinase activity of endogenous γPKC, leading to vulnerability of the expressed cells to oxidative stress (Lin et al., 2007). Verbeek et al. also reported that mutant (G118D, V138E, C142S) γPKC showed reduced phorbol ester-induced kinase activation at plasma membrane (Verbeek et al., 2008).
Loss of Purkinje cells in the PKCγ H101Y transgenic mouse
2009, Biochemical and Biophysical Research CommunicationsProtein kinase C epsilon activates lens mitochondrial cytochrome c oxidase subunit IV during hypoxia
2008, Experimental Eye ResearchCitation Excerpt :Because gap junctions are used in cell-to-cell communication pathways, PKCγ knock-out mice display learning deficits, insensitivity to pain, do not develop tolerance to alcohol like normal mice (Abeliovich et al., 1993a,b), and are more sensitive to hydrogen peroxide induced cataract formation (Lin et al., 2006). It is thought that these deficits are partially a result of the improper control of gap junctions due to loss of PKCγ (Lin et al., 2007). In contrast, PKCɛ is known to translocate to mitochondria during hypoxia in heart where it interacts with several targets.